PROJECT SUMMARY Persistent HIV-infected resting CD4+ T cells can remain undetected in tissue and organ reservoirs despite decades of successful antiretroviral therapy (ART). The lack of gene activity in resting CD4+ T cells enables the integrated replication-competent provirus to persist indefinitely. HIV replication in activated CD4+ T cells is cytopathic, and infected cells are cleared by the immune system. However, a small percentage of HIV-infected activated CD4+ T cells revert to the resting G0 phenotype and remain undetected in tissue reservoirs. These HIV- infected resting cells can be readily activated, and rebound viremia in patients with interrupted ART invariably originates from persistent HIV-infected tissue reservoirs. An ongoing project in my laboratory is focused on the essential role of heat shock protein 90 (Hsp90) in HIV replication. We have shown that mild heat shock (39.5C) accelerates HIV transcription in chronically infected T-cell lines and increases HIV replication up to 30-fold in primary human cells. Accelerated HIV transcription coincides with increased cellular Hsp90 activity at 39.5C. Hsp90 is responsible for activating cellular transcription factors, and the Hsp90 inhibitor 17-AAG significantly reduces gene expression by the NF-kB, NFAT, and STAT5 host transcription factors. Further, 39.5C reactivates HIV replication in ART-suppressed aviremic HIV-infected patient samples and in human resting CD4+ T cells isolated from fully suppressed humanized mice. Further, we show that a more potent next-generation Hsp90 inhibitor has even greater anti-HIV activity with a highly favorable therapeutic ranking and that clinical HIV subtypes display increased viral transcription at 39.5C in primary human T cells and macrophages. Most importantly, Hsp90 inhibition prevents HIV rebound in fully suppressed humanized mice, and we identified persistent HIV-infected human cells in the mouse spleen. Two different Hsp90 inhibitors blocked HIV transcription in this persistent tissue reservoir and sustained HIV remission up to 11 weeks after drug cessation. Further, we now have evidence that persistent HIV infection in spleen, brain, and lung reservoirs is established soon after inoculation, which produces infectious virus despite highly potent ART. HIV-infected human T cells and macrophages are also found in the mouse liver, bone marrow, and reproductive tract. We hypothesize that increased Hsp90 activity at 39.5C activates host transcription factors that are essential for HIV transcription. This hypothesis will be addressed in the following Specific Aims: 1) Determine the gene expression profile of heat-shock activated HIV-infected human resting CD4+ T cells, 2) Identify specific Hsp90 protein complexes that regulate HIV persistence in resting CD4+ T cells, and 3) Characterize persistent HIV reservoirs in vivo and investigate the source of rebound viremia. We anticipate these studies will lead to a better understanding of how Hsp90 regulates HIV transcription, and our in vivo results will provide insight into the nature of rebound viremia from persistent tissue reservoirs.